Antibodies are protein molecules belonging to a group of immunoglobulins generated by the immune system in response to an antigen. The structure of most antibody molecules is based on a unit comprising four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked together by disulphide bonds. Each of these chains is folded in discrete domains. The C-terminal regions of both heavy and light chains are conserved in sequence and are called the constant regions, comprising one or more so-called C-domains. The N-terminal regions of the heavy and light chains, also known as V-domains, are variable in sequence and determine the specificity of the antibody. The regions in the variable domains of the light and heavy chains (VL and VH respectively) responsible for antigen binding activity are known as the hypervariable or complementarity determining regions (CDR).
Immunoglobulins capable of exhibiting the functional properties of the four-chain immunoglobulins described above but which comprise two heavy polypeptide chains and which furthermore are devoid of light polypeptide chains have been described (WO 94/04678, Casterman et al, 1994). Fragments corresponding to isolated VH domains (hereinafter VHH) are also disclosed. Methods for the preparation of such antibodies or fragments thereof on a large scale comprising transforming a mould or yeast with an exoressible DNA sequence encoding the antibody or fragment are described in patent application WO 94/25591 (Unilever).
The immunoglobulins described in WO 94/04673, which may be isolated from the serum of Camelids, do not rely upon the association of heavy and light chain variable domains for the formation of the antigen-binding site but instead the heavy polypeptide chains alone naturally form the complete antigen binding site. These immunoglobulins, hereinafter referred to as “heavy-chain immunoglobulins” are thus quite distinct from the heavy chains obtained by the degradation of common (four-chain) immunoglobulins or by direct cloning which contribute part only of the antigen-binding site and require a light chain partner for antigen-binding, thus forming a complete antigen binding site.
Antibodies or fragments thereof, have found application in a variety of uses where the specific nature of the antibody-antigen interaction can be used to advantage. These include such uses as diagnosis, therapy, immunoassays and purification processes. The use of antibodies, or fragments thereof, in inhibiting viral infection has received attention, for instance during active immunisation with inactivated virus preparations or viral antigens produced in recombinant cells or during passive immunisation by the administration of neutralising antibodies.
It has been reported in the literature that monovalent Fab antibody fragments can neutralise viruses. Cheung et al (1992), Journal of Virology, 66, 6714–6720, describe the production of the Fab domain of a rabies virus-neutralising antibody MAb-57 and further demonstrate that this monovalent fragment itself has virus-neutralising activity. Other publications also report the capability of human Fab monovalent antibody fragments to neutralise or inhibit viral activity (see for example, Williamson et al (1993), Proc. Natl. Acad. Sci. USA, 90, 4141–4145). Such methods are not suitable for wide scale industrial application as the cost of producing such classical antibody fragments renders the processes economically unfeasible.
An alternative approach to inhibiting viral replication using antibodies which has been described in the literature is to select antibodies to target enzymes produced by the virus. Martin et al, Protein Engineering, 10(5), 607–614 (1997) describes the use of a camelisedf VH antibody fragment to inhibit hepatitis C virus MS3 protease, thereby preventing cleavage of the viral poly-protein precursor.
Another industrial application in which economically viable solutions to the problem of viral infection are sought is the field of fermentation processing, particularly food processing.
Lactic acid bacteria (LAB: Lactococci and Lactobacilli) play an important role in food fermentation processes such as the production of cheese or yoghurt. Often such fermentations are hampered by the sensitivity of the bacteria towards viruses, known as bacteriophage, which build up in these, often not aseptically performed, processes. A phage infection causes the LAB cells to lyse; during prolonged fermentations phage resistant cell populations can evolve, but this delay affects the production capacity severely, and the disturbed process yields a product of low quality. Sometimes the process has to be stopped prematurely, with complete loss of the batch of milk.
To date, the phage problem has mainly been approached by taking special precautions with respect to hygiene at the production facility, but this causes additional time delays. Another solution which has been proposed is the use of resistant LAB strains, but the regular appearance of adapted forms of bacteriophage forces the strains used to be changed from time to time in a procedure known as culture rotation. This has the disadvantage age of requiring labour intensive monitoring of the production facilities and medium for the presence of phage and requires the availability of several sets of cultures with the same functional attributes, differing only in phage sensitivity. There therefore remains considerable commercial interest in the further development of methods for combating LAB phage infection.
One method, proposed by Geller et al (1998), J. Dairy Sci., 81, 895–900, involves the use of colostrum from cows immunised with lactococcal phage as a source of phage-neutralising (polyclonal) antibodies to prevent lytic infection of Lactococcus lactis in fermentations of phage-contaminated milk. This method does provide a commercially viable solution to the problem, however. Not only is it extremely economically unattractive to produce antibodies in this way but furthermore, the addition of colostrum to milk does not have regulatory approval.
An alternative approach, which makes use of multivalent, multispecific antigen binding proteins comprising a polypeptide comprising in series two or more single domain binding units, preferably variable domains of a heavy chain derived from an immunoglobulin naturally devoid of light chains, to reduce the infectivity of LAB phages by cross-linking or agglutination is exemplified in the Applicant's co-pending patent application number PCT/EP98/06991, filed Oct. 26, 1998.
There remains a continuing need for the development of improved methods of inhibiting or neutralising viral infection. In particular, there remains continuing interest in development of methods which can be applied economically on a scale appropriate for industrial use.